Distinct shared and compartment-enriched oncogenic networks drive primary versus metastatic breast cancer

Metastatic breast-cancer is a major cause of death in women worldwide, yet the relationship between oncogenic drivers that promote metastatic versus primary cancer is still contentious. To elucidate this relationship in treatment-naive animals, we hereby describe mammary-specific transposon-mutagenesis screens in female mice together with loss-of-function Rb, which is frequently inactivated in breast-cancer. We report gene-centric common insertion-sites (gCIS) that are enriched in primary-tumors, in metastases or shared by both compartments. Shared-gCIS comprise a major MET-RAS network, whereas metastasis-gCIS form three additional hubs: Rho-signaling, Ubiquitination and RNA-processing. Pathway analysis of four clinical cohorts with paired primary-tumors and metastases reveals similar organization in human breast-cancer with subtype-specific shared-drivers (e.g. RB1-loss, TP53-loss, high MET, RAS, ER), primary-enriched (EGFR, TGFβ and STAT3) and metastasis-enriched (RHO, PI3K) oncogenic signaling. Inhibitors of RB1-deficiency or MET plus RHO-signaling cooperate to block cell migration and drive tumor cell-death. Thus, targeting shared- and metastasis- but not primary-enriched derivers offers a rational avenue to prevent metastatic breast-cancer.


Reviewer #2 (Remarks to the Author): Expert in breast cancer functional genomics
The authors of this manuscript performed Sleeping-beauty mutagenesis in a murine model of breast cancer with loss of Rb to identify genes that drive primary or metastatic tumor growth or both. Using this approach thy identified the MET-RAS pathway as shared driver, while Rho signaling, ubiquination and RNE processing is metastasis-only driver. The authors followed up more detailed characterization of selected screen hits including Fbxw7 and Pten by generating double knock out mice or knock down in human breast cancer cell lines by shRNA.
The mouse model MMTV-Cre:Rbf/f mice has been previously described, Sleeping-beauty mutagenesis screens have also been performed for mammary tumors in mice. Thus, novelty is limited, and the genes/pathways identified have well known roles in breast tumorigenesis.
The manuscript is very dense (both the text and content) making it hard to read.
Specific comments: 1. Mammary tumors in mice are virtually always hormone receptor negative basal subtype, mostly reflecting one subset of triple-negative breast cancer (TNBC).
2. Rb loss is not that common in human breast tumors, it mainly occurs in a subset of TNBC or treatment-resistant ER+ luminal tumors (especially if treated with CDK4/6 inhibitors).
3. If the lung tumors are true metastases, then they should be derived from the primary mammary tumors and therefore should be clonally related -including shared transposon mutations. On the other hand, if the Rb deleted tumors are metastatic on their own w/o any additional mutation, then this is not really a screen to identify drivers of metastasis, but just drivers of tumor growth.
4. The follow up validation experiments using selected genes did not actually show enhanced metastasis with any of the genes tested, just increased tumor growth. Thus, they did not really identify any metastasis drivers.
5. The mechanistic studies are limited to cell growth and migration/invasion assays, which are very limited superficial characterization of cellular phenotypes w/o giving any mechanistic insights.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): Expert in in vivo transposon mutagenesis screening and cancer functional genomics In this study by Jiang et al, the authors perform a mammary specific Sleeping Beauty (SB) mutagenesis screen to investigate the relationship between oncogenic drivers that promote metastatic versus primary breast tumors. The screen was performed in treatment-naïve animals and in the context of Rb loss of function, which is frequently lost in breast cancers. The investigators identified common insertion site (CISs) in primary tumors (P-drivers), metastatic tumors (M-drivers), and in primary and metastatic tumors (shared-S drivers). The screen is very well-done, and the work is extensive, however most of the identified genes and pathways are not novel. Multiple Sleeping Beauty breast cancer screens have previously been published. As the investigators did not emphasize the novelty of their results and distinguish their work from previously published SB screens, the current study is not well suited for publication in Nature Communications. Major and minor points should be addressed and will improve the overall study: Response: We thank the Reviewer for suggesting that we emphasize the novelty of our results. Our results introduce several novel concepts and advances as summarized below: -To our knowledge, this is the first Sleeping Beauty (SB) mutagenesis screen in the mammary gland/breast cancer conducted on both primary tumors and metastases. All previous breast cancer screens were done only on primary lesions. This is a major leap forward as metastatic disease is the major cause of death from breast cancer. -Another novelty of our work is the identification of oncogenic networks that drive metastatic breast cancer, and the demonstration of primary (P)-only, metastasis (M)-only and shared (S) oncogenic drivers that promote both primary and metastatic mammary tumors. -Critically, we showed that a similar pattern of primary-enriched, metastasis-enriched and shared oncogenic drivers is observed in human breast cancer (4 different data sets). We found Rho signaling/motility as a major metastatic driver that is enriched both in our metastatic SB screen and in human metastatic breast cancer. -TNBC patients with RB loss-MET-high (two S-drivers) or RB loss -RhoA high (S-driver plus M-driver) pathway activation exhibit exceedingly poor prognosis and should be prioritized for therapy. -Inhibitors of S-drivers (RB-loss, MET) plus M-driver (Rho pathway) cooperate to suppress cell proliferation and migration. -Our analysis supports the novel idea that S-drivers cooperate with compartment specific Por M-drivers to promote local versus distal growth, respectively, -Our results also suggest that "targeting S-and M-but not P-derivers offers a rationale avenue to prevent metastatic breast-cancer. " -we ended the revised Abstract with this sentence. -Our SB screens reveal metastasis-specific drivers even in drug-naïve animals. Thus, new mutations found in metastasis-only breast cancer samples from human patients may not only be due to drug selection but also represent genuine metastasis-promoting drivers. 2. It is stated on p.3 (line 124-126) that no report has demonstrated clonal relationships between primary and metastatic gCISs. This might be true for breast cancer, however this type of analysis has been performed in other Sleeping Beauty screens for different tumor types (liver cancer), for example in Keng et al. Nature Biotechnology, 2009. The authors should clarify/modify this statement. Response: We thank the Reviewer for pointing out this oversight on our part. We referenced the Keng et al. paper in the initial submission but did not credit it for demonstrating clonal relationship between primary and metastatic HCC. We have now revised the text as following ".. 3. In the current study, the authors claim that RhoA and PI3K pathways are elevated in metastatic versus primary lesions, representing metastasis-enriched oncogenic pathways. In contrast, many studies suggest that RhoA is involved in all stages of tumor progression. The authors should acknowledge this discrepancy and speculate/provide commentary regarding why RhoA signaling is specific to breast cancer metastasis. Response: We agree that Rho and PI3K signaling are elevated in primary tumors vs normal cells but our results show that these pathways are further and significantly induced in metastasis. The new statistical analysis on combined 4 cohorts (new Fig. 7b) clearly demonstrate that. We added the following statement in Discussion (p15) to clarify this point "Both Rho GTPase and PI3K signalings promote primary tumor proliferation and survival (Ref: 108-109), but our results reveal that these pathways are elevated in metastases and are thus ideal for therapeutic targeting together with S-drivers".
Minor points: 1. Figure 5 g,h,i are all mis-labeled as Figures 6 g,h,i (page 10, lines 423-425). Response: Thanks-we fixed thatto new Fig. 5h-j. 2. The following sentence is repeated twice on p. 12, "Induction of RhoA signalling in metastatic breast cancer could be caused by other genetic and epigenetic alternations, post-translational modification, or by external cues from the tumor microenvironment; their identification would be instrumental for precision therapy". Lines 541-544 and 545-547. Response: Thanks -we removed this duplication.

Reviewer #2 (Remarks to the Author): Expert in breast cancer functional genomics
The authors of this manuscript performed Sleeping-beauty mutagenesis in a murine model of breast cancer with loss of Rb to identify genes that drive primary or metastatic tumor growth or both. Using this approach they identified the MET-RAS pathway as shared driver, while Rho signaling, ubiquitination and RNA processing are metastasis-only drivers. The authors followed up more detailed characterization of selected screen hits including Fbxw7 and Pten by generating double knock out mice or knock down in human breast cancer cell lines by shRNA. The mouse model MMTV-Cre:Rbf/f mice has been previously described, Sleeping-beauty mutagenesis screens have also been performed for mammary tumors in mice. Thus, novelty is limited, and the genes/pathways identified have well known roles in breast tumorigenesis. The manuscript is very dense (both the text and content) making it hard to read. Response: We thank the Reviewer for suggesting that we emphasize the novelty of our results. Our results introduce several novel concepts and advances as summarized below: -To our knowledge, this is the first Sleeping Beauty (SB) mutagenesis screen in the mammary gland/breast cancer conducted on both primary tumors and metastases. All previous breast cancer screens were done only on primary lesions. This is a major leap forward as metastatic disease is the major cause of death from breast cancer.

-Another novelty of our work is the identification of oncogenic networks that drive metastatic breast cancer, and the demonstration of primary (P)-only, metastasis (M)-only and shared (S) oncogenic drivers that promote both primary and metastatic mammary tumors. -Critically, we showed that a similar pattern of primary-enriched, metastasis-enriched and shared oncogenic drivers is observed in human breast cancer (4 different data sets). We found Rho signaling/motility as a major metastatic driver that is enriched both in our metastatic SB screen and in human metastatic breast cancer. -TNBC patients with RB loss-MET-high (two S-drivers) or RB loss -RhoA high (S-driver plus M-driver) pathway activation exhibit exceedingly poor prognosis and should be prioritized for therapy. -Inhibitors of S-drivers (RB-loss, MET) plus M-driver (Rho pathway) cooperate to suppress
cell proliferation and migration.

-Our analysis supports the novel idea that S-drivers cooperate with compartment specific Por M-drivers to promote local versus distal growth, respectively, -Our results also suggest that "targeting S-and M-but not P-derivers offers a rationale avenue to prevent metastatic breast-cancer. " -we ended the revised Abstract with this sentence. -Our SB screens reveal metastasis-specific drivers even in drug-naïve animals. Thus, new
mutations found in metastasis-only breast cancer samples from human patients may not only be due to drug selection but also represent genuine metastasis-promoting drivers. -Finally, we show that the M-drivers form specific interactomes (Fig. 2a) and pathways (Fig.  3a), and that components of these interactomes/pathways correlate with poor clinical outcome ( Fig. 3c; supplemental Figs. S2, S3, S5). Thus, the Shared and Metastasis-specific drivers we have identified (Fig. 1, supplemental Excel Tables) provide a rich resource for future basic and translational analysis. We emphasized all these points in the revised manuscript, e.g. Abstract, last sentence; Discussion, first paragraph.

Finally, we made multiple changes in the manuscript to simplify the text, as requested.
Specific comments: 1. Mammary tumors in mice are virtually always hormone receptor negative basal subtype, mostly reflecting one subset of triple-negative breast cancer (TNBC).

Response: We discussed this issue with Chuck Perou, a co-author on this manuscript, who is an expert in breast cancer and molecular subtyping of human breast cancer and mouse models.
Virtually all mouse models are ER-negative, but not all are of the basal subtype. The following two references: PMID: 17493263 and PMID: 24220145 from Perou's lab, show that most mouse models are not basal-like subtype as determined by molecular profiling. Although mouse models are ER-negative/ER-low, ER level is just one of many possible 'luminal tumor' markers, and the widely used MMTV-NEU and MMTV-PYMT are classified as luminal but are ERnegative/low. There do exist human tumors that are luminal and ER-negative/PR-negative, most would call these TNBC "LAR" subtype as these TNBC luminal tumors are often AR+.
Finally, the Perou and Zacksenhaus labs showed that Rb-negative lesions from our MMTV-Cre:Rbf/f mouse model are diverse, and cluster together with luminal, basal-like or mesenchymallike/claudin-low like lesions (PMID: 20679727; PMID: 27571409).
2. Rb loss is not that common in human breast tumors, it mainly occurs in a subset of TNBC or treatmentresistant ER+ luminal tumors (especially if treated with CDK4/6 inhibitors). Response: We thank the Reviewer for pointing out this issue, which we have now further clarified in the manuscript by adding a sentence and the references below in the Introduction.
As indicated in the manuscript, RB can be disrupted by specific mutations/ deletions/epigenetic silencing of the gene or by phosphorylation of pRB via cyclin-dependent kinases. Phosphorylation dissociates pRB from its major targets such as E2F1-3, allowing cell cycle progression. The Perou's lab/Cancer Genome Atlas Network showed in a 2012 Nature paper (PMID: 23000897) that the RB gene is mutated in 2% of luminal and 4% of basal-like BC (Fig. 1,  ibid). However, the RB pathway is lost in ~20% of basal-like breast cancer through multiple additional mechanisms (e.g. RB mutation/silencing; cyclin E amplification; p16ink4a lost - Table 1, ibid). Using RB-loss/p53-loss signatures and oncoprint analysis of RB1 and TP53 alterations, we calculated that the two genes are lost in 28-40% of TNBC (JCI, 2016 -PMID: 27571409). Thus, while RB loss by nonsynonymous mutation is not that common, it is frequently inactivated by other mechanisms. Similar conclusion was presented in Nik-Zainal, S. et al (Nature, 2016; PMID: 27135926, Fig 1) in which RB1 loss is among the 9 most frequently altered genes in BC and second most altered in ER-negative BC. Another strategy for identification of cancer driver genes based on nucleotide context rather than nonsynonymous mutation (Nat Genet. 2020; PMID: 32015527) pointed to RB (Fig 4) and RB pathway (Fig. 6)  In this manuscript, conditional Rb deletion in the mammary gland mimics the different mechanisms that inactivate the Rb gene (mutations/deletions/silencing) or pRb protein (hyperphosphorylation) -and identifies oncogenic alterations that cooperate with such dysregulation of the cell cycle/Rb pathway to promote primary and metastatic disease. We conveyed this point on page 3 in the Introduction, and referenced the above papers on the frequency of RB1 loss in breast cancer.
3. If the lung tumors are true metastases, then they should be derived from the primary mammary tumors and therefore should be clonally relatedincluding shared transposon mutations. On the other hand, if the Rb deleted tumors are metastatic on their own w/o any additional mutation, then this is not really a screen to identify drivers of metastasis, but just drivers of tumor growth. Response: We suggest that because we were able to demonstrate clonality (Fig. 2b-d), the lung lesions represent true metastases and not independent primary lung tumors. Without demonstrating such clonal relationship, one cannot assume that "the lung tumors are true metastases".
Regarding the second point, although the Rb pathway as well as TP53 and the PI3K pathways are often altered in metastases of diverse types of malignancies including breast cancer (PMID: 28783718), each alteration alone is not sufficient to promote primary and metastatic disease. They require cooperating oncogenic events, and our screen has identified such cooperating oncogenic drivers in the context of RB loss that promote both primary growth and metastasis.
4. The follow up validation experiments using selected genes did not actually show enhanced metastasis with any of the genes tested, just increased tumor growth. Thus, they did not really identify any metastasis drivers. Response: We thank the reviewer for raising this issue. In the revised manuscript, we present new data to address this critique. Specifically, we determined the consequences of CDC42BPA and MTMR3 knock-down on both primary tumor growth and lung metastasis following orthotopic transplantation. We observed significant increase in lung metastasis in both cases (new Fig. 5i-j).
We note that we also show that M-drivers promote cell proliferation and/or migration when over-expressed or depleted in TNBC cells. Increased cell migration is metastasis-promoting activity and a hallmark of metastatic dissemination.

5.
The mechanistic studies are limited to cell growth and migration/invasion assays, which are very limited superficial characterization of cellular phenotypes w/o giving any mechanistic insights. Response: We thank the reviewer for raising this issue as well. In the revised manuscript, we present new data to address he mechanism by which CDC42BPA, a M-driver, affects cell migration. Specifically, we determined the effect of CDC42BPA-depletion alone or together with the ROCK inhibitor Fasudil on phosphorylation of Myosin Light Chain 2 (MLC2) at Thr18/Ser19, which controls contractility, and on cell migration. We found that CDC42BPA modulates MLC2 phosphorylation and cell migration by antagonizing the effect of ROCK in TNBC cells ( Fig. 5f and supplementary Fig. S7de). We note that for CDC42BPA and other representative M-drivers, we demonstrated they promote cell proliferation, migration and/or tumorigenesis and metastasis (new Fig. 5i-j); for FBXW4 we also identified several factors (BCl2 and MRPL37) as potential targets.